Project Summary Developments in genetic analysis technologies, particularly DNA sequencing, have been transformative to biomedical research. In contrast to genomic information, the barrier to accessing proteomic information, particularly enzyme activity, is dramatically higher. As aberrant enzyme activities are consistently observed in disease, this information is critical for appropriate diagnosis and treatment. In addition, the ability to probe the function of enzyme activities using chemical inhibitors is critical to our understanding of biology and disease. DNA-encoded chemical libraries (DELs) have emerged as a new tool that enables medicinal chemists to capitalize on the power of genetic techniques. The in vitro selection is the key process by which the function of the synthetic molecules in a DEL are encoded with DNA sequence populations. Inspired by this transduction of functional information of abiotic molecules, this project seeks to further exploit these capabilities into new areas. Our research program centers on advancing DNA-encoded chemical approaches for the development of chemical probes and for proteome-wide enzyme activity detection. This involves developing new approaches for the synthesis and selection of DNA-encoded chemical libraries, as well as the implementation of a new, related technology for DNA-based detection of enzyme activities using the in vitro selection of DNA-encoded proteomic probes. For chemical probe development, we have chosen two target classes that involve protein:protein interactions mediated through a short linear motifs: protein kinases and histone targeting domains. We will design and build DELs that target the protein substrate site of protein kinases to develop both selective inhibitors and non-natural substrates. Present use of ATP-competitive kinase inhibitors has been significantly limited by poor selectivity. Similarly, the overlapping selectivities of peptide substrates has limited their use in activity detection. Our targets for kinase protein substrate-competitive inhibitors and substrates are the Src family of tyrosine kinases. Also, we will develop inhibitors to the trimethyllysine peptide binding site of chromodomains in the CBX family of histone targeting proteins. The homology of the chromodomains in the eight chromobox (CBX) proteins and the nature of their binding site has made the development of inhibitors difficult. Selective probes generated here will be used to the decipher roles of CBX proteins in transcriptional regulation. With regards to activity detection, we will specifically address two enzyme families: kinases and serine hydrolases. We will develop a new approach to profile the activities of protein kinases by selection of DNA-linked substrates and DNA sequencing. We will implement this approach in studies to characterize the changes in activity that occur in the development of resistance to the kinase inhibitor lapatinib and also in the cellular process of epithelial- mesenchymal transition. In the next area, we will use selection of DNA-linked, activity-based probes to assess activity of the serine hydrolase family. We will develop a high throughput, multiplexed assay system for profiling libraries of chemical inhibitors across this entire enzyme family.